Duchenne muscular dystrophy (DMD) is a lethal disease that affects one in every 3500 males in the United States, causing severe disabilities and death. Despite the capacity of satellite cells to endogenously regenerate skeletal muscle, intramuscular transplantation efforts have shown very limited success due to poor cell survival, low retention, and suboptimal engraftment. Skeletal muscle consists of highly striated cells arranged in a highly anisotropic manner, attached to an extracellular matrix (ECM) composed of many fibers of feature sizes in the nanometer range, extending for various length scales with high fidelity. Our previous studies suggest that providing an ECM-based support for cells, mimicking the native muscle ECM nanotopography, can provide a physiologically relevant microenvironment for the stem cells to align and fuse together to form mature muscle. We also identified and demonstrated the role of sphigosine 1-phosphate (S1P) as a potent angiogenic and myogenic factor. The overall goals of this proposal are to develop nanopatterned, anisotropic 3D muscle tissue patches that closely mimic native skeletal muscle structure and then to test their ability to integrate and restore muscle function in a mouse model of DMD. We will combine a nanotopography-based tissue engineering approach with cell sheet manipulation techniques and S1P therapy to generate scaffold free 3D patches of vascularized muscle tissues. Primary muscle endothelial and satellite cells expressing Pax7Cre/flox GCaMP3 will be cultured on nanotopographically-defined, thermoresponsive substrates with tunable geometries to promote myogenesis and create anisotropic cell sheets. These sheets will be stacked to form 3D muscle constructs that will undergo molecular, structural, and functional analyses prior to transplantation. We will test our 3D tissue construct's ability to integrate with dystrophic muscle provide a stem cell reservoir, restore dystrophin expression and strengthen limb muscles in muscular dystrophy mice. Specific aims include: (1) to develop vascularized 3D muscle patches using nanopatterned cell sheet manipulation techniques; and (2) to implant and evaluate 3D muscle patches with sphingosine 1-phosphate conjugated fibrin to restore muscle function in a mouse model of Duchenne muscular dystrophy.